555 



Identification of formalin-preserved eggs of 

 red sea bream iPagrus major) (Pisces: Sparidae) 

 using monoclonal antibodies 



Shingo Hiroishi 



Yasutaka Yuki 



Eriko Yuruzume 



Faculty of Biotechnology 



Fukui Prefectural University 



1-1 Gakuen-cho 



Obama City, 917-0003 Fukui, Japan 



E-mail address (for S Hiroishi), hiroishi@fpu.ac.|p 



Yosuke Onishi 



Tomoji Ikeda 



Hironobu Komaki 



Kansai Environmental Engineer Center 

 1-3-5 Azuchi-cho, Chuo-ku 

 Osaka City, 541-0052 Osaka, Japan 



Muneo Okiyama 



Ocean Research Institute 

 University of Tokyo 

 1-15-1 Minamidai, Nakano-ku, 

 Tokyo, Japan 



Catches of important commercial fish 

 such as red sea bream, fiat fish, and 

 yellowtail are decreasing in Japan. 

 In order to sustain these species it is 

 especially important that their distri- 

 bution and biomass at all life stages 

 are known. However, information on 

 the early life stages of these species is 

 limited because identifying the eggs 

 and larvae of such fish is sometimes 

 extremely difficult. 



Mito (1960, 1979) and Ikeda and 

 Mito (1988) developed methods for 

 identifying pelagic fish eggs based on 

 morphological features. However, their 

 methods have limitations because 

 many unidentified eggs have similar 

 features. In addition, eggs are usu- 

 ally fixed in formaldehyde solution 

 just after collection in the field. This 

 procedure may alter several egg char- 

 acteristics and therefore prevent iden- 

 tification (Ikeda and Mito, 1988), or 

 make identification difficult when the 

 egg diameter measures 0.8-1.0 mm 

 because so many kinds of eggs fall in 

 that range. Thus, an alternative iden- 

 tification method would be useful. 



Effective genetic analyses for iden- 

 tifying fish eggs or larvae (or both) 

 have been developed by Graves et al. 

 (1989), Daniel and Graves (1994), 

 and Shao et al. (2002). However, 

 their methods may have limitations 

 if samples are preserved in formal- 

 dehyde for several years or if DNA 

 must be extracted from numerous 

 samples. In addition, we are lacking 

 the DNA sequences for many species 

 sequences that are necessary for iden- 

 tifying eggs in the field. 



We have successfully produced 

 monoclonal antibodies to differenti- 

 ate harmful marine phytoplankton 

 species from morphologically simi- 

 lar harmless species (Hiroishi et 

 al., 1988; Nagasaki et al., 1991b; 

 Sako et al., 1993; Vrieling et al., 

 1993; Hiroishi et al., 2002) as well 

 as Microcystis, a toxic fresh water 

 bloom-forming cyanobacteria (Kondo 

 et al., 1998). These antibodies were 

 obtained from a culture supernatant 

 solution of hybridoma cells that was 

 produced by a cell fusion procedure 

 between myeloma cells and antibody- 



producing spleen cells. The specific 

 antibodies described above could be 

 used to detect and quantify harmful 

 bloom-forming microorganisms that 

 react with the monoclonal antibod- 

 ies and that secondarily react with 

 fluorescein isothiocyanate conjugated 

 goat anti-mouse Ig(G+M) antibody. 

 With fluorescence microscopy with 

 B-exciting light, yellowish fluorescein 

 coronas around the cells of the toxic 

 species were observed, confirming a 

 positive reaction. These antibodies 

 can recognize different molecules 

 distributed on the cell surface, even 

 when the organisms have similar 

 morphological features. One of the 

 molecules distributed on Chattonella 

 was determined to be glycoprotein 

 (Nagasaki et al., 1991a). This method 

 would help us to differentiate small 

 marine organisms like fish eggs. 



Red sea bream (Pagrus major) 

 (Table 1) eggs can easily be distin- 

 guished from those of other sparids 

 also found in Japan, such as Acan- 

 thopagrus latus, by differences in egg 

 size and spawning seasons, and from 

 those of Evynnis japonica by differ- 

 ences in spawning seasons (Ikeda 

 and Mito, 1988; Kinoshita, 1988; 

 Hayashi. 2000). However, eggs of 

 some sparids, such as Aeanthopagrus 

 schlegeli, Sparus sarba, and Dentex 

 tumifrons are extremely difficult to 

 distinguish from eggs of P. major. 

 Therefore, we developed monoclonal 

 antibodies that allow P. major eggs 

 to be clearly identified by immuno- 

 staining, thus differentiating them 

 from other similar sparids. 



This technique may be a useful 

 new tool for identifying fish eggs. 

 Here, we report a method for identi- 

 fying P. major eggs using monoclonal 

 antibodies developed to react specifi- 

 cally with the eggs. 



Materials and methods 



Eggs of P. major were obtained from 

 adult female fish that had spawned in 



Manuscript submitted 4 April 2003 

 to Scientific Editor's Office. 



Manuscript approved for publication 

 2 March 2004 by the Scientific Editor. 



Fish. Bull. 102:555-560 (20041. 



